Methods and Compositions for Inhibition of ATR and FANCD2 Activation

ABSTRACT

This invention is announcing a composition of flavonoid skeleton in the formula I or formula II compound, wherein each of the substituents is given the definition as set forth in the specification and claims. This composition has the capacity to treating or preventing a virus infection in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/714,109, which was filed on Dec. 13, 2012 and claimedbenefit of Taiwanese Patent Application No. 101117920, which was filedon May 18, 2012. All of the above applications are incorporated byreference herein as if fully set forth.

The sequence listing titled “Sequence Listing” filed May 7, 2019 havinga file size of 1,850 bytes, and created on May 7, 2019, is incorporatedherein by reference as if fully set forth.

FIELD OF THE INVENTION

The application is related to the modulation of host cell factorsrequired for DNA and RNA virus infections. Specifically, the applicationis related to benzopyran-4-one derivative compounds that target humanhost cell factors involved in DNA and RNA virus infections, and the useof such compounds to modulate DNA and RNA virus infections and asantiviral agents.

BACKGROUND OF THE INVENTION

The benzopyran-4-one derivative compound of formula I has a flavonoidmoiety and was previously isolated and identified from the whole plantextract of Thelypteris torresiana, a fern species native to Taiwan.

The exposure and biological effects of Protoapigenone (I-1),5′,6′-Dihydro-6′-methoxy-protoapigenone (I-2) and Protoapigenin (I-3)compounds have been investigated by cytotoxicity assay. It was foundthat Protoapigenone (I-1) demonstrated therapeutic effects and was alead compound for potential anticancer drug development.

Furthermore, for developing a new potent anticancer drug, severalanalogues of I type compound such as I-4, I-5, I-6, I-7 and I-8, andanother II type moiety such as II-1 and II-2 compound are alsosynthesized or semi-synthesized. The compound I-4 having the chemicalname 2-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-4H-chromen-4-one can beexpressed by the general name of protoflavonone. The compound I-5 isalso termed as 5-hydroxyprotoflavone, and the chemical name is2-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-5-hydroxy-4H-chromen-4-one. Thecompound I-6 has the chemical name5-hydroxy-2-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-7-methoxy-4H-chromen-4-onewhich can be expressed by the general name of5-hydroxy-7-methoxy-protoflavonone. The homologous compounds I-4 and I-7have similar structures but a different function group on R11 positionsonly, which is a hydroxyl group and another is a methoxyl group in thatposition. The compound I-5, I-8, II-1 and II-2 also present the modifiedfunction group of R11 position models.

Formula II has an β-naphthoflavone moiety, and the compound II-1 has thechemical name3-(1-hydroxy-4-oxo-cyclohexa-2,5-dienyl)-1H-benzo[f]chromen-1-one.Compound II-2 has the chemical name 1′-methoxy-β-naphthoflavone.

In previous studies, Protoapigenone (I-1) and its more potent analogcompound II-1 (FIG. 1A) were shown to induce oxidative stress,consequently activating the p38 and JNK1/2 MAPK pathways following cellcycle arrest and apoptosis in several cancer cell types. These compoundswere also found to reduce the size of tumor xenografts in nude micewithout exerting toxic effects on the recipient. Recently, thosecompounds of formula I and II were found to induce chromosomal breakagethrough oxidative stress, implicating a role for benzopyran-4-onederivatives of formula I and II in interfering with DNA metabolism.Until now, the biomolecular actions and implications of thisbenzopyran-4-one derivative mediated interference are mostlyundetermined. Herein, we found that benzopyran-4-one derivatives arecapable of inhibiting DNA damage-induced activation of ATR targets Chk(Cell Cycle Checkpoint Kinase) 1 and FANCD2, which then sensitize tumorcells to chemotherapy, and finally results in tumor size reduction inmice.

During virus infection, the immune system of the host is typicallyactivated for defense. However, once it enters into a host cell, thevirus will exploit some of the host cell's machinery to replicate itselfat high speed. Several types of viruses have shown this replication,including the DNA viruses Epstein-Barr virus (EBV), herpes simplex virus1 (HSV-1), adenovirus and SV40, papilloma virus, Hepatitis B virus,sindbis virus, and the lentivirus human immunodeficiency virus (HIV),which lead to the activation of host DNA-damage response pathways. Theactivation of cellular DNA repair and recombination enzymes isbeneficial for viral replications. Further, research shows that newsmall-molecule inhibitors of the DNA-damage response pathways may be ofvalue to treat viral infections.

Strategies for identifying targets for antiviral intervention typicallyfocus on compounds that attack the viral proteins including thestructural components of the virion as well as viral genome-encodedenzymes which are necessary for propagation of the virus. The approachof targeting viral proteins has several limitations: i) the limitednumber of viral targets; ii) viral targets tend to be highly specific toa particular virus or even strain of virus; and iii) viruses are able torapidly alter their genetic composition to develop resistance toantiviral drugs. Another approach in antiviral drug development is todesign drugs to strengthen the host's factors to fight the viralinfection, rather than to fight the viral infection itself.

Our results show that these benzopyran-4-one derivatives compounds arenoteworthy potential to treat virus infection by the inhibition of ATRsignaling cascades.

SUMMARY OF THE INVENTION

The application is related to methods of treating DNA and RNA virusinfections and methods of treating or preventing a symptom or diseaseassociated with DNA and RNA virus infection, comprising administering toa subject a composition comprising a benzopyran-4-one derivativecompound that targets a human host cell factor involved in DNA and RNAvirus infections.

The present application is based, in part, on the discovery that virusinfections can be reduced by pharmacologically targeting human host cellfactors required for viral replications. Targeting host cell factors,rather than the viral factors required for influenza virus replication,may greatly reduce the emergence of viral resistance and expand thenumber of targets for antiviral intervention.

Disclosed herein are benzopyran-4-one derivative compounds that targethuman host cell factors involved in virus infection and replication.Disclosed herein are pharmaceutical compositions, comprising suchcompounds, and methods of using such compounds to modulate the virusinfection and replication. In some embodiments, the compounds andcompositions comprising them reduce or inhibit virus infection andreplication. Disclosed herein are methods of using such compounds andcompositions to reduce or inhibit virus infection and replication. Insome embodiments, the compounds modulate virus infection and replicationby altering the expression of, e.g., mRNA or protein and/or activity ofthe human host cell factors (e.g., ATR and FANCD2) involved in the virusinfection and replication. In some embodiments, the compounds reduce orinhibit virus infection and replication by reducing or inhibiting theexpression of mRNA or protein and/or activity of the human host cellfactors involved in the virus infection and replication. In someembodiments, the human host cell factor interacts with a component ofthe DNA and/or RNA viruses. In some embodiments, the human host cellfactor is required for the virus infection and replication.

The compounds disclosed herein, and for use in the compositions andmethods disclosed herein, target human host cell factors involved in DNAand RNA viruses and modulate DNA and RNA viruses. In some embodiments,the compounds disclosed herein, and for use in the compositions andmethods disclosed herein, target human host cell factors involved in DNAand RNA viruses and reduce or inhibit DNA and RNA viruses. The targetedhuman host cell factors may be required for DNA and RNA viruses. Thetargeted human host cell factors may be involved in or required for oneor more of the following events of the viral life cycles: entry;uncoating; nuclear import; viral RNA transcription; or viral RNAtranslation. The targeted human host cell factor may be involved in orrequired for replication of more than one strain of DNA and RNA viruses.For example, the human host cell factor may be involved in or requiredfor infection and replication of viruses including: double-stranded DNAviruses (Adenoviruses, Herpesviruses, Poxviruses, etc.); single-stranded(+)sense DNA viruses (Parvoviruses); double-stranded RNA viruses(Reoviruses and Birnaviruses); single-stranded (+)sense RNA viruses(Picornaviruses, Togaviruses, etc.); single-stranded (−)sense RNAviruses (Orthomyxoviruses, Rhabdoviruses, etc.); single-stranded(+)sense RNA viruses with DNA intermediates in the life-cycle(Retroviruses); and double-stranded DNA viruses with RNA intermediatesin the life-cycle (Hepadnaviruses).

In some embodiments, a compound disclosed herein, and for use in thecompositions and methods disclosed herein, targets a component orregulator of, or factor that interacts with, one or more of thefollowing categories of human host cell factors: cytoskeleton;ribonucleoprotein; spliceosome; ubiquitin/proteasome system; ribosome orother translation machinery; kinase; phosphatase; signaling (e.g.,G-protein coupled receptors and signaling at the plasma membrane);mitochondrion or mitochondrial ribosome; plasminogen; stress response;v-ATPase; ion channel or other ion transport; nucleus; sumoylation;nuclear transport; nucleotide binding; cell cycle; vesicular transport(e.g., COPI vesicle); chromosome; carboxylic acid metabolism; DNA damageresponse; or DNA repair. In some embodiments, a compound disclosedherein, and for use in the compositions and methods disclosed herein,targets a component or regulator of, or a factor that interacts with,one or more of the following categories of human host cell factors:ATR-Chk1 pathway; ATM-Chk2 pathway; MRN (Mre11-Rad50-Nbs1) complex;histone H2AX; MCPH1/BRIT1; CTIP; SMC1; IP3-PKC pathway; COPI vesicles;endosomal uptake, maturation, acidification, and fusion; actinorganization and function; PI3K-AKT pathway; endosomal recyclingpathway; MAPK pathway; proteases; calcium/calmodulin system; nucleartrafficking; trafficking; sumoylation; microtubule organization(including assembly) and function; autophagy; and ubiquitination.

In some embodiments, a compound disclosed herein, and for use in thecompositions and methods disclosed herein, targets a component orregulator of, or factor that interacts with, one or more of thefollowing categories of human host cell factors: the base excisionrepair proteins UNG, SMUG1, MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1,NEIL2 and NEIL3 (DNA glycosylases); APE1 or APEX2 (AP endonucleases);LIG1 or LIG3 (DNA ligases I and III); XRCC1 (LIG3 accessory); PNK orPNKP (polynucleotide kinase and phosphatase); PARP1, PARP2(Poly(ADP-Ribose) Polymerases); PolB or PolG (polymerases); FEN1(endonuclease) and Aprataxin.

In some embodiments, a compound disclosed herein, and for use in thecompositions and methods disclosed herein, targets a component orregulator of, or factor that interacts with, one or more of the baseexcision repair proteins PARP1, PARP2, and PolB. In other embodiments,the base excision repair protein is PARP1 or PARP2.

Yet another embodiment provides use of benzopyran-4-one derivativecompounds as a single agent (monotherapy) for treating virus. In someembodiments, the benzopyran-4-one derivative compounds are used to treatpatients having a virus infection in combinational therapy. In otherembodiments, said combinational therapy uses at least two compositionformulas of the composition.

In some embodiments, the compound is an agent that reduces or inhibitsthe expression of mRNA or protein and/or activity of a human host cellfactor involved in virus infection and replication. In some embodiments,the compound reduces or inhibits the interaction of a human host cellfactor with a component of the viruses. In some embodiments, thecompound reduces or inhibits one or more of the following events of theDNA and RNA viral life cycle: entry; uncoating; nuclear import; viralRNA transcription; and viral RNA translation. In some embodiments, thecompound reduces or inhibits replication of more than one strain of DNAor RNA viruses. For example, the compound may reduce or inhibitinfection and replication of viruses, such as double-stranded DNAviruses (Adenoviruses, Herpesviruses, Poxviruses, etc.); single-stranded(+)sense DNA viruses (Parvoviruses); double-stranded RNA viruses(Reoviruses and Birnaviruses); single-stranded (+)sense RNA viruses(Picornaviruses, Togaviruses, etc.); single-stranded (−)sense RNAviruses (Orthomyxoviruses, Rhabdoviruses, etc.); single-stranded(+)sense RNA viruses with DNA intermediates in the life-cycle(Retroviruses); and double-stranded DNA viruses with RNA intermediatesin the life-cycle (Hepadnaviruses).

In accordance with an aspect of the present invention, compounds ofbenzopyran-4-one derivatives, characteristically with inhibiting of DNADamage Response (DDR), are provided. The benzopyran-4-one derivativesincludes a common structure being the following formula I:

wherein: R₃, R₅, R₇, R₁₁, R₁₄ and R₁₆ are selected independently from agroup consisting of a hydrogen, a hydroxyl group, a methoxyl group and aoxygen atom contain a double bond.

In accordance with a further aspect of the present invention, compoundsof benzopyran-4-one derivatives, characteristically with ATR-mediatedDNA damage checkpoint, is provided. The benzopyran-4-one derivativesincludes a common structure being the following formula II;

wherein: R₂₁ is selected independently from a group consisting of ahydrogen, a hydroxyl group and a methoxyl group.

In a further aspect of the present invention, a method for assaying astate of DNA DDR kinase signaling cascades is provided. The methodincludes steps of:

-   -   providing a reaction site thereof;    -   adding to the reaction site an effective amount of a        benzopyran-4-one    -   derivative represented by one of formula I and formula II,

-   -   wherein each of R₃, R₅, R₇, R₁₁, R₁₄, and R₁₆ is one selected        from a group consisting of a hydrogen, a hydroxyl group, a        methoxyl group and a oxygen atom containing a double bond. R₂₁        is one selected from a group consisting of a hydrogen, a        hydroxyl group and a methoxyl group.

In a further aspect of the present invention, a method for treating orpreventing a virus infection in a subject is provided. The methodcomprises a step of administering to the subject with the virusinfection a therapeutically effective amount of a benzopyran-4-onederivative for a administration duration between 1 to 10 days to inhibita viral replication in the subject.

In a further aspect of the present invention, a method for inhibitingreplication of a virus in a subject is provided. The method comprises astep of administering to the subject at risk of developing a viralinfection a therapeutically effective amount of a benzopyran-4-onederivative at an interval selected from a group consisting of aonce-daily interval, a multiple-daily interval and a weekly interval.

In a further aspect of the present invention, a method for dealing witha virus infection in a subject, comprising steps of identifying thesubject with the virus infection; and administering to the subjectinfected by a virus a therapeutically effective amount of abenzopyran-4-one derivative.

The above objects and advantages of the present aspects will become morereadily apparent to those ordinarily skilled in the art after reviewingthe following detailed descriptions and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one color drawing orphotograph as a drawing executed in color. Copies of this patent orpatent application publication with color drawing(s) will be provided bythe Office upon request and payment of the necessary fee.

FIG. 1 illustrates Protoapigenone (I-1) induce chromosome aberration butdoes not produce marked DDR.

FIG. 2 illustrates immunoblots showing DDR by detecting phosphorylationof cell marker following exposure of HEK293T cell to 10 μM compound I-1for the indicated times.

FIGS. 3(a)-3(b) show that dose-dependent effects of compound I-1 andcompound II-1 on the inhibition of UV-induced Chk1 phosphorylation incells.

FIG. 3(a) illustrates immunoblots showing the expression of MDA-MB-231cell.

FIG. 3(b) illustrates immunoblots showing the expression of A549 cell.

FIG. 4 shows the cytotoxic effect of compound against cell line.

FIGS. 5(a)-5(b) show benzopyran-4-one derivatives inhibit DNAdamage-induced DDR.

FIG. 5(a) illustrates immunoblots showing the expression of inhibitingChk1 phosphorylation.

FIG. 5(b) illustrates immunoblots showing the expression of A549 cell.Cells are pretreated with 50 μM okadaic acid (OA) or 20 μM MG132 andsubjected to 10 J/m² UV for 1 h to induce DDR.

FIGS. 6(a)-6(b) show benzopyran-4-one derivatives inhibit UV-inducedChk1 phosphorylation.

FIG. 6(a) illustrates immunoblots showing the expression of A549 cell.

FIG. 6(b) illustrates immunoblots showing the expression of MDA-MB-231cell. Cells are pretreated with chemicals for 20 min and subjected to 10J/m² UV for 1 h to induce DDR.

FIG. 7 shows benzopyran-4-one derivatives inhibit chemotherapeuticagents-induced Chk1 phosphorylation.

FIGS. 8(a)-8(b) show benzopyran-4-one derivatives inhibit ATR-dependentChk1 phosphorylation.

FIG. 8(a) illustrates immunoblots showing the expression DDR induced byH₂O₂ (peroxide).

FIG. 8(b) illustrates immunoblots showing the expression DDR induced byhydroxyurea (HU).

FIG. 9 shows the effect of compound II-1 on phosphorylation were assayedafter UV irradiation on HEK293T cell, and this exhibited decreasedexpression of genes following the RNA interference treatment.

FIGS. 10(a)-10(c) show benzopyran-4-one derivatives inhibit UV- orH₂O₂-induced Chk1 phosphorylation.

FIG. 10(a) illustrates immunoblots showing compound I-1 inhibitH₂O₂-induced Chk1 phosphorylation.

FIG. 10(b) illustrates immunoblots showing compound I-1 inhibitUV-induced Chk1 phosphorylation.

FIG. 10(c) illustrates immunoblots showing compound II-1 inhibitH₂O₂-induced Chk1 phosphorylation.

FIG. 11 illustrates immunoblots showing compound I-1 inhibitH₂O₂-induced Chk1 phosphorylation. Effects of compound I-1 on Chk1phosphorylation were assayed 1 h after 10 J/m² UV irradiation on HEK293Tcell overexpressing ATRIP, TopBP1, claspin, or ATR following delivery oftagged full-length cDNA constructs for 48 h.

FIGS. 12(a)-12(b) show benzopyran-4-one derivatives inhibit DNA damagecheckpoint and repair.

FIG. 12(a) illustrates percentages of the mitotic marker.

FIG. 12(b) illustrates the expression FACS (Fluorescence Activated CellSorting) dot blot for analyzing the percentage of GFP cell denoting theHRR frequency.

FIG. 13 illustrates the percentage of M-phase cells with γH2AX foucusformation.

FIGS. 14(a)-14(c) show benzopyran-4-one derivatives inhibitcisplatin-induced Chk1 phosphorylation and FANCD2 monoubiquitination.

FIG. 14(a) illustrates immunoblots showing the effect oncisplatin-induced DDR in A594 cell.

FIG. 14(b) illustrates immunoblots showing the effect oncisplatin-induced DDR in U2OS cell.

FIG. 14(c) illustrates immunoblots showing the effect oncisplatin-induced DDR in MDA-MB-231 cell.

FIGS. 15(a)-15(b) show in vitro clonogenic survival for A549 cell.

FIG. 15(a) illustrates the effects fraction of compound I-1.

FIG. 15(b) illustrates the effects fraction of compound II-1.

FIGS. 16(a)-16(b) show in vitro clonogenic survival for MDA-MB-231 cell.

FIG. 16(a) illustrates the effects fraction of compound I-1.

FIG. 16(b) illustrates the effects fraction of compound II-1.

FIGS. 17(a)-17(b) show chemosensitization effect of benzopyran-4-onederivatives.

FIG. 17(a) illustrates the effects fraction of compound I-1.

FIG. 17(b) illustrates the effects fraction of compound II-1.

FIG. 18 shows in vivo xenograft tumor volume for MDA-MB-231 cell.

-   -   A—cisplatin 2 mg/kg    -   B—cisplatin+compound II-1 0.2 mg/kg    -   C—compound II-1 0.2 mg/kg

FIGS. 19(a)-19(b) show optical activity of benzopyran-4-one derivatives.

FIG. 19(a) illustrates the effects on A549 cells

-   -   A—control    -   B—0.25 μM compound I-1    -   C—0.5 μM compound I-1    -   D—1 μM compound I-1

FIG. 19(b) illustrates the effects on MDA-MB-231 cells

-   -   A—control    -   B—0.25 μM compound I-1    -   C—0.5 μM compound I-1    -   D—1 μM compound I-1

FIGS. 20(a)-20(b) show optical activity affected by benzopyran-4-onederivatives concentration.

FIG. 20(a) illustrates the effects of compound I-1.

-   -   A—control    -   B—0.1 μM compound II-1    -   C—0.2 μM compound II-1    -   D—0.4 μM compound II-1

FIG. 20(b) illustrates the effects of compound II-1.

-   -   A—control    -   B—0.1 μM compound II-1    -   C—0.2 μM compound II-1    -   D—0.4 μM compound II-1

FIGS. 21(a)-21(b) show cell cycle progression rate.

FIG. 21(a) illustrates the effects of unsynchronized cells.

FIG. 21(b) illustrates the effects of control group.

FIGS. 22(a)-22(c) show that rate of cell cycle progression were treatedwith benzopyran-4-one derivatives for 6 hrs.

FIG. 22(a) illustrates the effects of control group.

FIG. 22(b) illustrates the effects of compound I-1.

FIG. 22(c) illustrates the effects of compound II-1.

FIGS. 23(a)-23(c) show that rate of cell cycle progression were treatedwith benzopyran-4-one derivatives for 9 hrs.

FIG. 23(a) illustrates the effects of control group.

FIG. 23(b) illustrates the effects of compound I-1.

FIG. 23(c) illustrates the effects of compound II-1.

FIGS. 24(a)-24(e) show number of labeled DNA replication.

FIG. 24(a) illustrates the effects of DMSO group.

FIG. 24(b) illustrates the effects of Hydroxyurea (HU) group.

FIG. 24(c) illustrates the effects of ku55933 group.

FIG. 24(d) illustrates the effects of compound I-1.

FIG. 24(e) illustrates the effects of compound II-1.

FIG. 25 illustrates the percentage of EdU incorporation.

FIG. 26 illustrates the treatment protocol for virus infection andcompound treatment.

FIG. 27(a) illustrates the effects of pre-treatment of the compounds onthe number of GFP cells.

FIG. 27(b) illustrates the effects of pre-treatment of the compounds onthe mean fluorescence intensity (MFI) of the GFP protein inAd-GFP-infected cells.

FIG. 27(c) illustrates the effects of pre-treatment of the compounds onthe MFI of the GFP protein in Lenti-GFP-infected cells.

FIG. 28(a) illustrates the effects of post-treatment of the compounds onthe number of GFP cells.

FIG. 28(b) illustrates the effects of post-treatment of the compounds onthe MFI of the GFP protein in Ad-GFP-infected cells.

FIG. 28(c) illustrates the effects of post-treatment of the compounds onthe MFI of the GFP protein in Lenti-GFP-infected cells.

FIG. 29(a) illustrates the effects of pre-treatment of the compounds onthe number of GFP cells.

FIG. 29(b) illustrates the effects of pre-treatment of the compounds onthe MFI of the GFP protein in Ad-GFP-infected cells.

FIG. 29(c) illustrates the effects of post-treatment of the compounds onthe number of GFP cells.

FIG. 29(d) illustrates the effects of post-treatment of the compounds ofthe MFI of the GFP protein in Ad-GFP-infected cells.

FIG. 30 illustrates the effects of the treatments of the compounds onthe survival rate.

FIG. 31(a) illustrates the surviving fraction of MDA-MB-231 cells whichare co-administered compound II-1 with doxorubicin.

FIG. 31(b) illustrates the surviving fraction of MDA-MB-231 cells whichare co-administered compound II-1 with etoposide.

FIG. 31(c) illustrates the surviving fraction of MDA-MB-231 cells whichare co-administered compound II-1 with mytomycin C.

FIG. 31(d) illustrates the surviving fraction of MDA-MB-231 cells whichare co-administered compound II-1 with camptothecin.

FIG. 31(e) illustrates the surviving fraction of MDA-MB-231 cells whichare co-administered compound II-1 with taxol.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Further embodiments herein may be formed by supplementing an embodimentwith one or more element from any one or more other embodiment herein,and/or substituting one or more element from one embodiment with one ormore element from one or more other embodiment herein.

The phrase “virus infection” in this disclosure refers to DNA virusinfection, RNA virus infection or both of them, and the term “virus” inthis disclosure refers to DNA virus, RNA virus or both of them.

EXAMPLES

The following non-limiting examples are provided to

illustrate particular embodiments. The embodiments throughout may besupplemented with one or more detail from one or more example below,and/or one or more element from an embodiment may be substituted withone or more detail from one or more example below.

Ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) are 2members of the phosphoinositide 3-kinase (PI3K)-related protein kinasesfamily that play a central role in DNA damage response (DDR)coordination; they also function in the signaling cascades machinery ofcell cycle arrest, DNA repair and transcription, and cell death. WhileATM is predominant activated in response to DNA strand breaks, ATR isactivated in response to damage arising from ultraviolet (UV) ray orreplication block; both kinases activate signaling cascades thatinvolving 2 checkpoint kinases effectors, Chk1 and Chk2, whose roleswere previously suggested to be redundant. In contrast to ATM, ATR hasbeen reported to be indispensable for cell growth and for life.ATR-knockout mouse embryos died early due to mitotic catastrophecharacterized by incomplete DNA replication and chromosomalfragmentation. Moreover, ATR gene mutations are rarely found in humans.The only mutated variants that can survive are heterozygous orhypomorphic variants. Furthermore, cells derived from patients withSeckel syndrome exhibit cellular features associated with ATR signalingcascades defects. Consistent with this phenotype, seckel-like mouseembryonic cells showed accelerated aging due to replicative stress,exhibiting an accumulation of lethal chromosomal breaks. However, withregard to its role in regulating the replication checkpoint, ATR isactivated by most cancer chemotherapeutic agents that target DNA inreplicating cells. Therefore, inhibition of ATR signaling cascades is avalid and promising strategy that can improve chemotherapeutic orradiotherapeutic efficiency.

Thus so far, several inhibitors of DDR-related kinases, including Chk1and Chk2, have been successfully used alone or in combination with eachother in clinical trials. Recently, several chemicals that inhibit ATRkinase activity in vitro were used to support the hypothesis that ATRkinase can be targeted to improve cancer therapy. Since most of thesestudies are in their initial stages, it is imperative to focus moreefforts toward investigating strategies to inhibit ATR signalingcascades.

In accordance with an aspect of the present invention, benzopyran-4-onederivatives compound, characteristically with inhibiting of DNA DamageResponse (DDR), is provided.

Another aspect of this invention, pharmaceutical composition ofbenzopyran-4-one derivatives, characteristically with inhibiting of DNADamage Response (DDR), is provided. The benzopyran-4-one derivativesincludes a common structure being the following formula I or formula II,

-   -   wherein: R₃, R₅, R₇, R₁₁, R₁₄ and R₁₆ are selected independently        from a group consisting of a hydrogen, a hydroxyl group, a        methoxyl group and a oxygen atom contain a double bond. R₂₁ is        selected independently from a group consisting of a hydrogen, a        hydroxyl group and a methoxyl group.

In a further aspect of this invention is directed towards a method oftreating cancer in a subject in need thereof, including the sequentialor simultaneous co-administration of a compound of benzopyran-4-onederivatives or a pharmaceutically acceptable salt thereof, and aDNA-damaging agent. In some embodiments, said DNA-damaging agent isselected from chemotherapeutic drugs such as alkylating agents,antimetabolic agents, antibiotic anti-cancer agents, Topoisomeraseinhibitors and anti-mitosis agents.

In some embodiments, said alkylating agent is one selected from Nitrogenmustards (e.g. Melphalan, mechlorethamine, Chlorambucil, Ifosfamide,Cyclophosphamide, Estramustine and phenoxybenzamine); or Aziridines(e.g. Thiotepa, Carboquone); or Nitrosoureas (e.g. Carmustine,Semustine, Iomustine, Nimustine, Streptozocin, Ranimustine andLomustine); or Procarbazine and triazenes (e.g. Dacarbazine,Temozolomide and Procarbazine); or Alkyl sulfonate (e.g. Busulfan); orPlatinum coordination complex (e.g. Cisplatin, Carboplatin, Nedaplatin,Iproplatin and Oxaliplatin); and mixtures thereof.

In some embodiments, said antimetabolic agents are one selected fromThymidylate synthase inhibitor (e.g. Aminopterin, Methotrexate, Tegafur,Piritrexin, Trimetrexate, Floxuridine, Raltitrexed, Pemetrexed,Fluorouracil, Doxifluridine and Capecitabine); or Amidophosphoribosyltransferase inhibitors (e.g. Mercaptopurine, Thioguanine andThionosine); or DNA chain elongation inhibitors (e.g. Cytarabine,Ancitabine, Gemcitabine, Fludarabine, Cladribine, Clofarabine,Azaserine, Azacitidine, Pentostatin, Hydroxyurea); and mixtures thereof.

In some embodiments, said antibiotic anti-cancer agent is one selectedfrom free radical agents (e.g. Bleomycin and Actinomycin D); orTopoisomerase II inhibitors (e.g. Daunorubicin, Doxorubicin, Idarubicin,Epirubicin, valrubicin, Pirarubicin, Aclarubicin, MitoxantroneandPiroxanthrone); or other therapies or anticancer agents (e.g. Menogaril,Plicamycin, Acivicin, Anthramycin, Pentostatin, Calicheamicin andPeplomycin); and mixtures thereof.

In some embodiments, said Topoisomerase inhibitor is one selected fromTopoisomerase I inhibitors (e.g. Camptothecin, Irinotecan, Topotecan);or Topoisomerase II (e.g. Podophyllin, Podophyllotoxin, Etoposide,Teniposide); and mixtures thereof.

In some embodiments, said anti-mitosis agent is one selected fromPaclitaxel and Docetaxel; or anti-microtubule agents (e.g. Colchicine,Vinblastine, Vincristine, Vindesine and Vinorelbine); and mixturesthereof.

Benzopyran-4-one derivatives compounds of this invention includeProtoapigenone (I-1), 5′,6′-dihydro-6′-methoxy-protoapigenone (I-2),Protoapigenin, (I-3), protoflavonone (I-4), 5-hydroxyprotoflavone (I-5),5-hydroxy-7-methoxy-protoflavonone (I-6), compounds I-7, compounds I-8,3-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-1-H-benzo[f]chromen-1-one (II-1)and compounds II-2.

In a further aspect of this invention, pharmaceutical composition ofbenzopyran-4-one derivatives, characteristically with modulating theactivation state of ATM kinase is provided. The benzopyran-4-onederivatives includes a common structure being the following formula I orformula II,

In a further aspect of this invention, both assay kit and assaycomposition of benzopyran-4-one derivatives, characteristically withdetecting the activation state of ATR, DNA DDR kinase signaling cascadesis provided.

In a further aspect of this invention is directed towards a method ofanalyzing ATR, DNA DDR kinase signaling cascades in a reaction sitethereof, including the sequential or simultaneous of benzopyran-4-onederivatives compound or a pharmaceutically acceptable salt thereof, anda chemotherapeutic drugs or additional agent. In some embodiments, saidchemotherapeutic drug is selected from chemotherapeutic drugs such asalkylating agents, antimetabolic agents, antibiotic anti-cancer agents,Topoisomerase inhibitors and anti-mitosis agents.

In some embodiments, the individual components of the combination may beadministered separately, at different times during the course oftherapy, or concurrently, in divided or single combination forms. Alsoprovided is, for example, simultaneous, staggered, or alternatingtreatment.

In a further aspect of this invention, compounds and pharmaceuticalcomposition of benzopyran-4-one derivatives, characteristically withdetecting of DNA damage in cancer cell as determined by the activationstate of ATM kinase is also useful for monitoring therapeutic effectsduring treatment.

In some embodiments, method using benzopyran-4-one derivatives compoundor pharmaceutical composition for defecting in the ATR signaling cascadeand/or DNA-damage response (DDR). In some embodiments, said defect isaltered expression or activity of one or more of the following cellmarkers as determined by standard cell marker detection assays: ATM,CHK1, CHK2, cellular tumor antigen p53, Adenosine monophosphateactivated protein kinase (AMPK), mammalian target of rapamycin complex(mTORC) 1, metal response element (MRE) 11, mitogen-activated proteinkinase (MAPK), MAPK-activated protein kinase (MAPKAPK) 2, DNA RepairProtein (RAD50), Nijmegen breakage syndrome (NBS) 1, 53BP1, mediator ofDNA damage checkpoint (MDC) 1, H2A histone family member X (H2AX).

In another embodiment, the cell is a cancer cell expressing DNA damagingoncogenes. In some embodiments, said cancer cell has altered expressionor activity of one or more of the following cell markers as determinedby standard cell marker detection assays: K-Ras, N-Ras, H-Ras, Raf, Myc,Mos, E2F, Cdc25A, CDC4, CDK2, Cyclin E, Cyclin A and Rb.

In a further embodiment, the invention relates to an assay kit or assaycomposition for determent of ATR and/or DNA DDR signaling cascades atreaction site. In particular the assay kit or assay composition caninclude, a benzopyran-4-one derivatives compound, a processing/handlingplan, a compartment, a additional reagent and instructions for use, or areagent with a compartment and instructions for use. In one embodiment,for the purpose of altered expression or activity can then generate adetectable at the reaction site of the immunocomplex.

The additional reagent can include ATR, the ATR receptor, the complex ofDNA, or an antigenic fragment thereof, a binding composition, or anucleic acid. A kit for determining the binding of a test compound,e.g., acquired from a biological sample or from a chemical library, caninclude a control compound, a labeled compound, and a method forseparating free labeled compound from bound labeled compound and acombination thereof.

The term excipients or “pharmaceutically acceptable carrier orexcipients” and “bio-available carriers or excipients” above-mentionedinclude any appropriate compounds known to be used for preparing thedosage form, such as the solvent, the dispersing agent, the coating, theanti-bacterial or anti-fungal agent and the preserving agent or thedelayed absorbent. Usually, such kind of carrier or excipient does nothave the therapeutic activity itself. Each formulation prepared bycombining the derivatives disclosed in the present invention and thepharmaceutically acceptable carriers or excipients will not cause theundesired effect, allergy or other inappropriate effects while beingadministered to human. Accordingly, the derivatives disclosed in thepresent invention in combination with the pharmaceutically acceptablecarrier or excipients are adaptable in the clinical usage and in thehuman. A therapeutic effect can be achieved by using the dosage form inthe present invention by the local or sublingual administration via thevenous, oral, and inhalation routes or via the nasal, rectal and vaginalroutes. About 0.1 mg to 1000 mg per day of the active ingredient isadministered for the patients of various diseases.

The carrier is varied with each formulation, and the sterile injectioncomposition can be dissolved or suspended in the non-toxic intravenousinjection diluents or solvent such as 1,3-butanediol. Among thesecarriers, the acceptable carrier may be mannitol or water. Besides, thefixing oil or the synthetic glycerol ester or di-glycerol ester is thecommonly used solvent. The fatty acid such as the oleic acid, the oliveoil or the castor oil and the glycerol ester derivatives thereof,especially the oxy-acetylated type, may serve as the oil for preparingthe injection and as the naturally pharmaceutical acceptable oil. Suchoil solution or suspension may include the long chain alcohol diluentsor the dispersing agent, the carboxylmethyl cellulose or the analogousdispersing agent. Other carriers are common surfactant such as Tween andSpans or other analogous emulsion, or the pharmaceutically acceptablesolid, liquid or other bio-available enhancing agent used for developingthe formulation that used in the pharmaceutical industry.

The composition for oral administration adopts any oral acceptableformulation, which includes capsule, tablet, pill, emulsion, aqueoussuspension, dispersing agent and solvent. The carrier generally used inthe oral formulation, taking a tablet as an example, the carrier may belactose, corn starch and lubricant, and magnesium stearate is the basicadditive. The excipients used in a capsule include lactose and driedcorn starch. For preparing an aqueous suspension or an emulsionformulation, the active ingredient is suspended or dissolved in oilinterface in combination with the emulsion or the suspending agent, andappropriate amount of sweetening agent, flavors or pigment is added asneeded.

The nasal aerosol or inhalation composition may be prepared according tothe well-known preparation techniques. For example, the bioavailabilitycan be increased by dissolving the composition in the phosphate buffersaline and adding the benzyl alcohol or other appropriate preservative,or the absorption enhancing agent. The compound of the present inventionmay be formulated as suppositories for rectal or virginaladministration.

The compound of the present invention can also be administeredintravenously, as well as subcutaneously, parentally, muscular, or bythe intra-articular, intracranial, intra-articular fluid andintra-spinal injections, the aortic injection, the sterna injection, theintra-lesion injection or other appropriate administrations.

Protoapigenone (I-1) induces chromosomal aberrations but does notproduce marked DDR.

Previously, Protoapigenone (I-1) and compound II-1 were demonstrated tocause DNA strand breaks and apoptosis in lung and prostate cancers (ChenH M, et al., Free Radic Biol Med 2011), suggesting that inducing DNAdamage may be the potential mechanism underlying the anticancer effectof benzopyran-4-one derivatives.

To test this hypothesis, we investigated the cytogenetic effect ofProtoapigenone (I-1) on CHO cells (FIG. 1). According to the Table 1,low Protoapigenone (I-1) concentrations produced dose-dependentincreases in chromosomal structural changes, such as breakages, radials,and chromosomal polyploidy, similar to the effects seen with mitomycin Ctreatment; however, the complete mitotic chromosome could not beobtained upon high-dose Protoapigenone (I-1) treatment.

TABLE 1 Protoapigenone (I-1) induces chromosomal aberration in CHO cellsDMSO Protoapigenone Treatment control (I-1) Mitomycin C Concentration(μM) 0.00   2.17   4.35   2.00 Chromatid break (No.) 0 2 1 2 Chromatiddeletion 0 0 1 3 Triradial 0 4 13  42  Quadriradial 0 3 9 29  Ring 0 1 20 Complex rerrangement 0 0 0 2 Dicentric 0 0 0 1 Polyploid 1 3 1 0Pulverized cell 0 0 1 5 Average aberrant 0.5   6.5 *  14.0 *  42.0 *metaphases (%)^(a) Note: 1. Two hundred cells per treatment wereanalysized for chromosomal aberration. 2. Type of structuralaberrations, such as chromatid break, chromatid deletion, triradial,quadriradial, ring, complex rerrangement, dicentric, polyploid andpulverized cell numbers (No.) were indicated. 3. Others chromosome gap,chromosome break, chromosome deletion and chromatid gap were not beobserved in this experiment. 4. ^(a),* indicated statistic significantlyfor tested vs. control group by t-test.

Since mitomycin C can induce DDR in many cancers, we investigated whatkind of DDR signaling was activated by Protoapigenone (I-1).Surprisingly, high doses of Protoapigenone (I-1) in HEK293T cells didnot induce noticeable changes in the putative DDR signaling, which wemeasured by analyzing the phosphorylation of the ATM-dependent Chk2Thr⁶⁸ residue and the ATR-dependent Chk1 Ser³⁴⁵ residue (FIG. 2). We didobserve that Protoapigenone (I-1) treatment caused slight accumulationof the p53 protein, which could have been the result of severalposttranslational modifications. However, phosphorylation of the p53Ser¹⁵ residue did not contribute to this Protoapigenone (I-1)-inducedp53 protein accumulation, suggesting that Protoapigenone (I-1) does notdirectly damage DNA because DNA damage normally stimulatesATM/ATR-dependent p53 Ser15 phosphorylation. Our result is similar toprevious reports that p38 MAPK is activated by Protoapigenone (I-1)(Chen W Y, et al. Invest New Drugs 2011), as its downstream targetMAPKAPK2 was found to be phosphorylated starting as early as 2 h afterProtoapigenone (I-1) exposure (FIG. 2). We repeated the benzopyran-4-onederivatives experiment on lung and breast carcinoma cell lines A549 andMDA-MB-231 cells, respectively, and obtained similar results.Consistently, no marked changes in Chk1 and Chk2 phosphorylationsignaling were detected even at high doses of either drug for as long as8 h after drug treatment (FIGS. 3(a) and 3(b)). The cytotoxic effect bybenzopyran-4-one derivatives on cancer cells was determined by MTT assayat 48 h of incubation (FIG. 4); our data indicated that the IC₅₀ valuerange for cytotoxicity was similar to those in previous reports,confirming that benzopyran-4-one derivatives are stable compounds thatdo not directly cause DNA damage.

Protoapigenone (I-1) and compound II-1 inhibit Chk1 phosphorylationafter DNA damage.

Understanding the mechanism by which the benzopyran-4-one derivativescompounds cause chromosomal breakages (FIG. 1) and other abnormalitiesmight aid in identifying their targets. We hypothesized that genes withfunctions associated with DNA damage checkpoints and/or DNA repair mightbe targeted by benzopyran-4-one derivatives. To test this hypothesis, weassessed the effects of benzopyran-4-one derivatives on DDR induced byH₂O₂. Protoapigenone (I-1) was found to inhibit Chk1, but promote Chk2phosphorylation in A594 cells treated with 0.1 mM H₂O₂ for 2 h; however,ATM autophosphorylation was not affected (FIG. 5(a)). Pretreatment ofcells with okadaic acid (OA) (a phosphatase inhibitor) or MG132 (aproteasome inhibitor) could not reverse the Protoapigenone (I-1)-inducedinhibition of Chk1 phosphorylation, indicating that the inhibition doesnot occur due to phosphatase activation or proteasome degradation byother regulatory factors (FIG. 5(b)). Further, we investigated othersources of DNA stimuli specific for ATR activation; our resultsdemonstrate that UV-induced Chk1 phosphorylation was dose-dependentlyinhibited by benzopyran-4-one derivatives within different cells (FIGS.6(a) and 6(b)). In response to DNA double-strand breaks (DSBs), FANCD2is known to be monoubiquitinated on K561 (FANCD2-Ub) in an ATR-dependentmanner to stimulate repair (Andreassen P R, et al. Genes Dev 2004). Weshowed that FANCD2-Ub was also inhibited by benzopyran-4-one derivatives(FIGS. 5(a), 6(a) and 6(b)); further, ATR inhibition by benzopyran-4-onederivatives was also observed in cells treated with currently prescribedchemotherapeutic agents (FIG. 7). Collectively, these findings indicatethat benzopyran-4-one derivatives can modify ATR signaling after varioustypes of DNA damage. Interestingly, compound II-1 was more potent thanProtoapigenone (I-1) in inhibiting Chk1 phosphorylation and cytotoxicity(FIGS. 4, 6(a) and 6(b)).

We speculate that the replacement of 2 hydroxyl groups on Protoapigenone(I-1) with an additional benzene ring contributes positively to this ATRinhibition; however, the definite pharmacophores need to be furtherinvestigated when the ATR protein structure is resolved.

Target specificity of Protoapigenone (I-1) and compound II-1 forATR-mediated signaling inhibition.

To elucidate the specificity of the benzopyran-4-one derivativesinhibition on ATR-mediated signaling, we compared the change betweencells treated with benzopyran-4-one derivatives or the ATM-specificinhibitor KU55933 before the induction of DDR. After H₂O₂ damage, ATM isthought to be the principal responder, and KU55933 treatment stronglyinhibited ATM-mediated Chk2 phosphorylation specifically, but its effecton ATR-mediated Chk1 phosphorylation was small (FIG. 8(a)). In contrast,after hydroxyurea (HU; a replication blocker) damage, ATR is thought tobe the principal responder, and benzopyran-4-one derivatives treatmentsignificantly inhibited Chk1 phosphorylation, but only slightlyinhibited Chk2 phosphorylation (FIG. 8(b)).

Using these pharmacological methods, we demonstrated that thespecificity of DDR inhibition between benzopyran-4-one derivatives andKU55933 was completely different. To strengthen the argument thatbenzopyran-4-one derivatives specifically inhibits ATR signaling, smallinhibitory RNAs against ATM, ATR, and the catalytic subunit of DNAprotein kinase (DNA-PKcs) were introduced into HEK293T cells beforeexposure to UV or H₂O₂.

Our results demonstrated that benzopyran-4-one derivatives completelyinhibited UV-induced or H₂O₂-induced Chk1 phosphorylation in a mannersimilar to siRNA knockdown of ATR, but not ATM or DNA-PKcs (FIGS. 9,10(a), 10(b) and 10(c)). The siRNAs against ATM and DNA-PKcs decreasedthe UV-induced or H₂O₂-induced Chk2 phosphorylation, which were notaltered by the addition of Protoapigenone (I-1), but were increased bycompound II-1 treatment. Interestingly, neither siRNA targeted to ATM orATR nor DNA-PKcs affected the compound II-1-mediated increase in Chk2phosphorylation. Since a high dose of compound II-1 itself slightlyinduces Chk2 activation (FIGS. 3(a) and 3(b)), the increased Chk2phosphorylation was likely a synergistic effect due to DNA damage.

To further identify the specific mediator that contributes to the effectof Protoapigenone (I-1) on the initiation of ATR kinase activation, wetested whether TopBp1, ATRIP, and claspin were involved, as they havebeen identified as mediators of ATR kinase activation (Lopez-Contreras AJ, et al. DNA Repair (Amst) 2010). Our results demonstrated thatoverexpression of ATRIP or TopBP1 did not reverse the inhibitory effectof Protoapigenone (I-1) on Chk1 phosphorylation, whereas overexpressionof claspin or ATR did (FIG. 11), suggesting that Protoapigenone (I-1)might affect the function of ATR or claspin contributes to ATR signalinginhibition.

Protoapigenone (I-1) and compound II-1 impair the functions of DNAdamage checkpoints and DNA repair.

Previously, it has been demonstrated that S/M and G2/M checkpoints areactivated by ATR in response to different types of DNA damage (Nghiem P,et al. Proc Natl Acad Sci USA 2001). Of these, the G2/M checkpointinvolves ATM and ATR in collaboration, whereas the S/M checkpoint ismediated solely by ATR. To maintain genetic integrity, ATR can preventpremature mitotic entry in the event of incomplete DNA replication orunrepaired DNA damage. In order to evaluate the effect ofbenzopyran-4-one derivatives on these ATR-associated DNA damagecheckpoints, we observed the effect of benzopyran-4-one derivatives onmitotic entry following hydroxyurea or cisplatin treatment. InMDA-MB-231 cells, hydroxyurea and cisplatin significantly decreased thenumber of mitotic cells, indicating that the S/M and G2/M checkpointsare intact in MDA-MB-231 cells (FIG. 12(a)).

TABLE 2 The percentage of mitotic cells Compound Control group I-1 II-1KU55933 Control group 20.28 ± 0.86  23.69 ± 0.94  12.05 ± 0.68 20.29 ±1.48 Hydroxyurea 4.46 ± 0.67 3.40 ± 0.41 10.49 ± 1.17 11.19 ± 0.76Cisplatin 5.35 ± 0.07 7.99 ± 0.91  8.14 ± 0.99  9.70 ± 0.25

Benzopyran-4-one derivatives or KU55933 treatment increased thepercentage of mitotic cells in cisplatin-treated cells, as the Table 2suggesting that all of these compounds inhibited the damage-induced G2/Mcheckpoint. However, benzopyran-4-one derivatives, but not KU55933,significantly increased the HU-induced mitotic entry that is specificfor the S/M checkpoint, indicating that benzopyran-4-one derivativesspecifically impaired this distinctive checkpoint mediated solely by ATR(FIG. 12(a)).

ATR function is also linked to DNA repair via its coupled targets(Sorensen C S, et al. Nat Cell Biol 2005). To examine the effect ofbenzopyran-4-one derivatives treatment on DNA repair, we performed ahomologous recombination repair (HRR) assay in HeLa cells.

TABLE 3 The percentage of GFP cell (DNA homologous recombination repairassay) Treatment GFP cell (%) Un-treatment group 0.0 μM 0.040 ± 0.0097chromosomal breaks Un-treatment 0.0 μM 1.217 ± 0.0203 generated byI-SceI Compound I-1 2.0 μM 0.807 ± 0.0403 endonuclease 4.0 μM 0.213 ±0.0105 expression group Compound II-1 0.2 μM 0.703 ± 0.0304 0.4 μM 0.170.0169

Our result, as Table 3 demonstrated that chromosomal breaks normallyrepaired by HRR were dose-dependently inhibited by Protoapigenone (I-1)at low concentrations. Compound II-1 produced similar effects at dosesthat were 10-fold lower than that of Protoapigenone (I-1) (FIG. 12(b)).From these results, we assumed that the cells carrying unrepaired DNAwould enter into mitosis following DNA damage. To verify thisassumption, we analyzed the DNA-damage marker gamma-H2AX on mitoticcells using immunofluorescence staining. As expected, the numbers oflarge gamma-H2AX foci were increased upon addition of benzopyran-4-onederivatives in both unperturbed and perturbed mitotic cells (FIG. 13),suggesting that benzopyran-4-one derivatives increase DNA damage inmitotic cells. The chromosomes became flat and aggregated afterbenzopyran-4-one derivatives treatment, differing from thethree-dimensional and hair-like appearance of normal chromosomes atmetaphase.

Protoapigenone (I-1) and compound II-1 enhance chemosensitivity.

Inhibition of the checkpoint and repair mechanisms leads tochemosensitization in cancers. We questioned whether benzopyran-4-onederivatives could function as sensitizers for the chemotherapeutic drugscisplatin that has been shown to induce ATR activation as well as FANCD2monoubiquitination, which is the vital step for DNA crosslink repair(Chirnomas D, et al. Mol Cancer Ther 2006). We found thatbenzopyran-4-one derivatives treatment decreased the cisplatin-inducedChk1 phosphorylation and FANCD2 monoubiquitination in A549, MDA-MB-231,and U2OS cells (FIG. 14(a), 14(b) and 14(c)). Using the same doses,compound II-1 not only inhibits monoubiquitination of FANCD2 but alsoaffects FANCD2 protein stability; this data emphasizes that compoundII-1 has more potent inhibitory effects as compared to Protoapigenone(I-1). We further treated individual cells with cisplatin in combinationwith several varying doses of benzopyran-4-one derivatives, and countedsurvival colonies to determine their ability to survive aftercisplatin-induced damage. Our results demonstrated that benzopyran-4-onederivatives effectively decreased the clonogenic survival incisplatin-treated MDA-MB-231 and A549 cells in the nanomolar dose range(FIGS. 15(a), 15(b), 16(a) and 16(b)). To investigate thechemosensitization effect of low-dose benzopyran-4-one derivatives invivo, we established a tumor xenograft in nude mice using humanMDA-MB-231 tumor cells, which are considered to be more resistant tocisplatin and are also sensitive to treatment with benzopyran-4-onederivatives, at least as compared to A549 cells (FIGS. 4, 17(a) and17(b)). When the mice were treated with 0.2 mg/kg compound II-1 incombination with 2 mg/kg cisplatin, the tumor inhibitory effect wasgreater than that of cisplatin treatment alone (FIG. 18). However,Protoapigenone (I-1) unexpectedly did not affect the cisplatinsensitivity of MDA-MB-231 tumors when a higher dose of 2 mg/kg was usedin our experiments (data not shown). The pharmacokinetic data ofProtoapigenone (I-1) and compound II-1 needs to be compared in futurestudies to determine the differences in the chemical effects of these 2compounds in vitro and in vivo.

In addition, there is a selectivity of chemotherapeutic agents incombination with the benzopyran-4-one derivatives. Specifically, onlythe chemosensitivity of a specific chemotherapeutic drug can be enhancedby the benzopyran-4-one derivatives. The specific chemotherapeutic drugwhich can be enhanced by the benzopyran-4-one derivatives is a DNAdamaging agent which can induce DDR.

The DNA damaging agent includes a DNA-reactive agent, an antimetabolity,a topoisomerase poison or a combination thereof.

The DNA-reactive agent includes, but is not limited to, a nitrogenmustard, a nitrosourea, a triazene, a natural resource alkylating agentand a alkylating-like platinum agent. The nitrogen mustard includes, butis not limited to, DNA alkylators cyclophosphamide, chlorambucil, andmelphalan. The nitrosourea includes, but is not limited to, carmustine,lomustine, and semustine. The triazene includes, but is not limited to,dacarbazine, and temozolomide. The natural resource alkylating agentincludes, but is not limited to, mitomycin C and streptozotocin. Thealkylating-like platinum agent includes, but is not limited to,cisplatin, carboplatin, and oxaliplatin.

The antimetabolity includes, but is not limited to, a pyrimidine analog,a purine analog and a nucleotide synthesis blocker. The pyrimidineanalog includes, but is not limited to, 5-fluorouracil, capecitabine,floxuridine, and gemcitabine. The purine analog includes, but is notlimited to, 6-mercaptopurine, 8-azaguanine, fludarabine, and cladribine.The nucleotide synthesis blocker includes, but is not limited to,methotrexate, aminopterin, pemetrexed, and ralitrexed.

The topoisomerase poison includes a topoisomerase II poison, atopoisomerase I poison and an anthracycline with the topoisomerase IIpoison plus a DNA intercalating property. The topoisomerase II poisonincludes, but is not limited to, etoposide and teniposide. Thetopoisomerase I poison is camptothecin. The anthracycline with thetopoisomerase II poison plus the DNA intercalating property includes,but is not limited to, doxorubicin, daunorubicin, epirubicin, andidarubicin.

Please refer to FIGS. 31(a)-31(e), which are the surviving fractions ofMDA-MB-231 cells which were co-administered compound II-1 with the DNAdamaging agent, i.e. (a) doxorubicin, (b) etoposide, (c) mytomycin C,(d) camptothecin, or (e) taxol. In FIGS. 31(a)-31(c), when 2.5 nM to20.0 nM compound II-1 is co-administered with doxorubicin, etoposide ormytomycin C, it can be seen that the survival of MDA-MB-231 cells issignificantly decreased. However, the survival of MDA-MB-231 cells isnot significantly decreased when 2.5 nM to 20.0 nM compound II-1 isco-administered with camptothecin or taxol. Therefore, thebenzopyran-4-one derivatives are pharmaceutically effective incombination with doxorubicin (a TOP2 inhibitor), etoposide (a TOP2inhibitor), and mitomycin C (an alkylating agent causing thecross-linking of DNA), but less effective in combination withcamptothecin (a TOP1 inhibitor), and even no effect in combination withtaxol (a microtubule inhibitor), which is not a DNA damage agent.Accordingly, a low dose of benzopyran-4-one derivatives can enhance thechemosensitivity of the chemotherapeutic drug.

The Enhancement of the chemosensitivity of the specific chemotherapeuticdrug can increase its chemotherapeutic effect. In an embodiment, thebenzopyran-4-one derivative and the chemotherapeutic drug can beco-administered at the same time to treat a subject (such as a mammalsubject) who suffers from one of a recurrent cancer, a cancer having acancer chemotherapy resistance, a cancer having a resistance to DNAdamage response (DDR) and a combination thereof.

ATR are involved in DNA replication. Low doses of Protoapigenone (I-1)and compound II-1 significantly slowed cancer growth in a dose-dependentmanner (FIGS. 19(a), 19(b), 20(a) and 20(b)), and caused S phase delayand inhibition of DNA synthesis (FIGS. 22(a), 22(b), 22(c), 23(a), 23(b)and 23(c)); these events are similar to previously reportedcharacteristics of ATR defects. In the results of double-thymidine cellcycle synchronization assay, according to the Table 4 which sorted outfrom FIGS. 21-23, the unsynchronized cells (FIG. 21(a)) becomesynchronization by using this method, and 97% of cells were stopped atG1/S boundary after two cycles of thymidine blocks (FIG. 21(b)). Thosesynchronized cells released from thymidine blockade and allowed progressinto S phase in presence or absence of protoapigenone (I-1) and compoundII-1. Protoapigenone (I-1) (FIGS. 22(b) and 23(c)) and compound II-1(FIGS. 22(c) and 23(c)) showed significantly reduce the percentage ofG2/M cells at 9 hours of treatment in compared with control group (FIGS.22(a) and 23(a)). So far, indicating that benzopyran-4-one derivativeswith the ability to delay the S phase progression.

Through EdU (5-ethynyl-2′-deoxyuridine) incorporation to measurement thecapacity of DNA replication, 4 μM protoapigenone (I-1) (FIG. 24(d)) and0.4 μM compound II-1 (FIG. 24(e)) but not 10 μM ATM inhibitor KU55933(FIG. 24(c)) showed efficiently reduce the percentage of incorporationin compared with control group (FIG. 24(a)), indicating that DNAreplication is inhibited (FIG. 25); these events are similar to theeffect of a DNA replication blocker Hydroxyurea (HU) (FIG. 24(b)) andvisualized that benzopyran-4-one derivatives with the ability to inhibitthe DNA replication.

TABLE 4 Cell cycle G1 S G2M Unsynchronized cells 42.51% 30.26% 24.75%Initiation (0 hrs) Control 70.95% 24.00% 1.29% Release 6 hrs Control18.29% 75.63% 0.63% synchronizeation I-1 25.45% 69.13% 0.41% II-1 21.72%72.01% 4.59% 9 hrs Control 20.94% 25.53% 53.54% I-1 21.41% 60.89% 16.46%II-1 15.50% 63.06% 20.45%

Materials and Methods

Antibodies

Primary antibodies of Chk1 (sc-8408), Chk2 (sc-17747), FANCD2(SC-20022), phospho-ATM Ser1981 (sc-47739), and Myc (sc-40) werepurchased from Santa Cruz. Phospho-histone H3 Ser10 (06-570) and H2AXSer139 (05-636) antibodies were purchased from Millipore. Claspin (2880)and phospho-Chk1 Ser345 (2348), phospho-Chk2 Thr68 (2661), phospho-P53Ser15 (9286), P38 MAPK Thr180/Tyr182 (9216), and MAPKAPK2 Thr334 (3007)were purchased from Cell Signaling. Actin (A2066), flag (F1804), andhemagglutinin (H9658) antibodies were purchased from Sigma-Aldrich. ATR(A300-137A), ATRIP (A300-095A), and TopBP1 (A300-111A) antibodies werepurchased from Bethyl; and anti-ATM (GTX70103) antibodies were purchasedfrom Gene Tex.

Cell Culture and Treatment.

MDA-MB-231 (breast adenocarcinoma; ATCC HTB-26, BCRC 60425) and A549(lung adenocarcinoma; ATCC CCL-185, BCRC 60074) human cell lines werepurchased from Bioresource Collection and Research Center (BCRC,Hsinchu, Taiwan), and were authenticated by American Type CultureCollection (ATCC, Manassas, Va.). U2OS (osteosarcoma), HeLa (cervicaladenocarcinoma), and HEK 293T (embryonic kidney cells) human cell lineswere provided by Dr. Sheau-Yann Shieh (Institute of Molecular Biology,Academia Sinica, Taipei, Taiwan). Cells were maintained in Dulbecco'smodified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 10%fetal bovine serum (FBS) (Gibco). For DDR induction, freshly dilutedH₂O₂ (Merck) was added to the culture medium 1 h before the cells wereharvested. For UV irradiation treatment, the cells were irradiated for10 J/m² by a cross-linker (UVP) 1 h prior to analysis. Protoapigenone(I-1) and compound II-1 were isolated and synthesized as describedpreviously (15-17).

In Vitro Chemosensitization Assay.

To evaluate in vitro chemosensitization, cells were seeded in 6-wellplates 1 d before the experiment at a density of 100-400 cells/well. Thedrugs were incubated with the cells for 6 h, after which the medium wasreplaced with fresh drug-free FBS-containing medium. The colonies becamevisible and were counted 7-10 d later using 0.1% crystal violet stainingfollowing image capture by a CCD camera (LAS-4000 mini; Fujifilm).

Flow Cytometry.

To evaluate the effect of DNA damage checkpoint activation on cell cycledistribution, the cells were harvested at indicated time points andfixed with methanol for at least 2 h. The DNA was then stained with asolution containing propidium iodide (PI) and RNase A (Sigma-Aldrich).Fluorescently labeled cells were subsequently analyzed by the flowcytometer (LSR II; BD Biosciences) with a suitable selection ofexcitation and emission wavelengths. The percentages of differentfluorescent cell populations were analyzed using WinMDI Ver. 2.9 (TheScripps Research Institute).

DNA replication was measured using a Click-it EdU assay kit, which isbased on incorporation of the thymidine analogue5-ethynyl-2′-deoxyuridine (EdU) into DNA during replication(Invitrogen). Then, 10 μM EdU was added to the cell culture medium 30min before the cells were harvested and fixed in 4% paraformaldehyde.After cycloaddition, EdU was detected with Alexa Fluor 647 using clickreaction catalyzed by Cu (II), and the DNA content was stained byCellCycle 405-blue. To assay the mitotic entry, cells were treated withthe indicated drugs and trapped in 70 nM nocodazole for 16 h, andantibodies against phospho-histone H3 Ser10 and PI were used to stainthe mitotic cells and DNA content, respectively. An FITC Annexin Vapoptosis detection kit was used to characterize the phenotype of celldeath based on PI and Annexin V double staining (BD Pharmingen, SanDiego, Calif.). Fluorescence-labeled cells were subsequently analyzed bythe BD LSR II flow cytometer with a suitable selection of excitation andemission wavelengths. The percentages of different fluorescent cellswere analyzed using WinMDI Ver. 2.9.

In Vitro Chromosome Aberration Test

In brief, 5×10⁵ Chinese Hamster Ovary (CHO) cells were seeded in 60-mmdishes 1 d before the experiment. Protoapigenone (I-1)-inducedstructural chromosomal changes after 20 h were compared with that of thecells cultured in 2 μM mitomycin C. At 18 h after Protoapigenone (I-1),0.1 μg/mL colchicine was added for 2 h, and metaphase cells werecollected by shaking them off the dishes. Mitotic cells were treatedwith 0.5% KCl for 10 min and fixed with a 3:1 mixture of methanol:glacial acetic acid. The cells were then spread on slides for chromosomestaining with 5% Giemsa solution. We then analyzed the chromosomestructure of 200 well-spread metaphase cells (100 metaphasecells/experiment) under a 100× oil immersion objective.

Plasmids and siRNAs

The plasmids ATR, ATRIP, and claspin were kindly provided by Dr. X. Wu(The Scripps Research Institute, La Jolla, Calif.), and TopBP1 wasprovided by Dr. J. Chen (University of Texas MD Anderson Cancer Center,Houston, Tex.). The siRNA sequences of the target ATM(5′-AAGCGCCTGATTCGAGATCCT-3′) [SEQ ID NO: 1], ATR(5′-CCTCCGTGATGTTGCTTGATT-3′) [SEQ ID NO: 2], DNA-PKcs(5′-GATCGCACCTTACTCTGTTGA-3′) [SEQ ID NO: 3], and the random sequencethat served as the control (5′-AAGTCAATATGCGACTGATGG-3′) [SEQ ID NO: 4]were synthesized by Sigma-Proligo (23, 24). All transfections in HEK293Tcells were performed by the calcium phosphate precipitation method.

Western Immunoblotting

Cell lysate preparations, gel electrophoresis, and immunoblotting wereperformed as previously described (23). The binding of primaryantibodies were detected by horseradish peroxidase-coupled secondaryantibodies (Jackson ImmunoResearch) followed by enhancedchemiluminescence (ECL) (Millipore). The images of non-saturated bandswere captured using a luminescent image analyzer (LAS-4000 mini;Fujifilm). The antibodies used in this study are listed in supplementarymaterials.

DNA Homologous Recombination Repair Assay

DNA constructs of the recombination substrate pHPRT-DRGFP, in which theI-SceI site lies within 1 copy of 2 mutated tandem repeated GFP genes,and the I-SceI endonuclease expression vector pCBASceI, were originallyconstructed by Dr. M. Jasin (25). In brief, we generated a stablepHPRT-DRGFP construct in HeLa cells, and evaluated the chromosomalbreaks generated by I-SceI endonuclease expression. Six hours afterpCBASceI was delivered into the cells, complete medium with or withoutProtoapigenone (I-1) or compound II-1 was replaced onto the cells.Forty-eight hours after delivery, the efficiency of chromosomal HRR wasobtained as the percentage of GFP-positive cells, which was assessed byflow cytometry.

Human Xenograft Tumors in Nude Mice.

Human breast cancer MAD-MB-231 cells were harvested from the culture,resuspended in medium without serum at 1×10⁸ cells/mL, and 0.1 mL ofthis suspension was subcutaneously injected into the right flank offemale nude mice (BALB/cAnN-Foxn1nu/Crl Narl; purchased from theNational Science Council Animal Center, Taiwan). Tumor-injected micethat successfully developed tumors that grew to approximately 50-100 mm³in volume were randomly sorted into groups and used for the experiments.Control vehicle or 2 mg/kg of cisplatin with or without 0.2 mg/kg ofcompound II-1 was administered intraperitoneally every 4 d throughoutthe experiment.

Example 1: Preparation of the Composition in Tablet

Tablets are prepared using standard mixing and formation techniques asdescribed in U.S. Pat. No. 5,358,941, to Bechard et al., issued Oct. 25,1994, which is incorporated by reference herein in its entirety.

Protoapigenone (I-1) 100 mg Lactose qs Corn starch qs

Proteins that are involved in DNA DNA-damage response pathways arepositive for virus replication, including ATM, ATR, NBS, and FANCD2. Wefound that benzopyran-4-one derivative compounds can inhibit cells inresponse to oxidative stress, ultraviolet, and DNA-damagingchemotherapeutics induced signaling. Therefore, benzopyran-4-onederivative compounds may be able to inhibit virus infections byinhibiting the DNA damage signaling pathway.

To test the effects of anti-virus infection of benzopyran-4-onederivative compounds, a green fluorescent protein (GFP) expressingadenovirus to visualize adenovirus infection was used. To obtain thereporter virus, the GFP cDNA is amplified from pEGFP-N1 (Clontech,Accession: U55762.1) through a polymerase chain reaction with primers5′-CACCATGGTGAGCAAGGGC-3′ [SEQ ID NO: 5] and 5′-TACTTGTACAGCTCGTCCATG-3′[SEQ ID NO: 6], and then cloned into pENTR™/D-TOP vector (Invitrogen).The GFP cDNA was transferred to adenovirus vector pAd/CMV/V5-DEST™through an in vitro recombination method using LR Clonase® II after thesequence was confirmed. We selected the ampicillin- andchloramphenicol-resistant clones to obtain the pAd-GFP. The pAd-GFPplasmid is then transfected into E1a and E1b expressing HEK293A cells toproduce a crude GFP expression adenoviral stock (Ad-GFP). The adenoviruswas amplified by infecting HEK293A cells. The adenoviral stock wastitered and later used to infect the cells for the analysis.

In addition to the test of the benzopyran-4-one derivative compounds onthe DNA virus infection, they were tested on the retrovirus using aGFP-expressing retrovirus to visualize the adenovirus infection. Toobtain this virus, plasmids (obtained from the National RNAi CoreFacility at Academia Sinica, Taiwan.) packaging plasmid pCMV-ΔR8.91(containing gag, pol and rev genes), envelope plasmid pMD.G (VSV-Gexpressing plasmid), and TRC library plasmid pLKO_AS3.1w.eGFP.neo (GFPcDNA carrying plasmid) were co-transfected into a 6-well plate of thelarge T antigen expressing package cell line, HEK293T cells. 24 hoursafter transfection, BSA-containing media per plate was replaced with theoriginal media to increase virus production, and the supernatantlentivirus was collected after an additional 16 hours. Afterward, thelentiviral stock (Lenti-GFP) was titered and used to infect the cellsfor analysis.

To observe the anti-virus effect of the benzopyran-4-one derivativecompounds, Ad-GFP and Lenti-GFP at a low MOI of 0.5 were then applied toinfect the HEK293A cells and HEK293T cells, respectively. The protocolsfor virus infection and compound treatment were shown in FIG. 26. In theprevention group, cells were treated by compounds of Compound I-1 at0.2, 1, and 2 μM or Compound II-1 at 0.1, 0.5, 1 μM for 30 minutesbefore Ad-GFP and Lenti-GFP infection. In the treatment group, cellswere treated by compounds of Compound I-1 at 0.2, 1, and 2 μM orCompound II-1 at 0.1, 0.5, and 1 μM for 2 hours after Ad-GFP andLenti-GFP infection. After a total of 6 hours of compounds and virustreatments, cells were washed with Hank's buffered salt solution and aflash culture media was replaced.

24 hours after virus infection, the effect of the benzopyran-4-onederivative compounds on the antivirus infection was recorded byobserving the GFP positive cells under an inverted fluorescencemicroscope. The mean fluorescence intensity by flow cytometry in eachplate of cells was analyzed for quantification.

According to the results, pre-treatment of 0.1-1 μM of Compound II-1decreased the number of GFP cells (FIG. 27(a)) significantly with adose-dependent effect, as well as the mean fluorescence intensity (MFI)of GFP protein (FIG. 27(b)) in Ad-GFP-infected cells. Pre-treatment ofCompound I-1 for 0.2-2 μM did not prevent the Ad-GFP infection. Theseresults indicate that Compound II-1 is more potent in prevention ofadenovirus infection than Compound I-1. Pre-treatment of 0.2-2 μM ofCompound I-1 or 0.1-1 μM of Compound II-1 significantly decreased theMFI of GFP protein (FIG. 27(c)) in Lenti-GFP-infected cells with adose-dependent effect. These results indicate that the benzopyran-4-onederivative compounds prevent the retrovirus infection. Compound II-1 ismore potent to prevent retrovirus infection than Compound I-1.

Post-treatment of 0.1-1 μM of Compound II-1 after 2 hours of Ad-GFPvirus infection decreased the number of GFP cells significantly with adose-dependent effect (FIG. 28(a)), as well as the MFI of GFP protein(FIG. 28(b)) in Ad-GFP-infected cells. Treatment with Compound I-1 for0.2-2 μM did not inhibit the Ad-GFP infection. These results indicatethat Compound II-1 is more potent in treating adenovirus infection thanCompound I-1. Post-treatment of 0.1-1 μM of Compound II-1 after 2 hoursof Lenti-GFP infection significantly decreased the MFI of GFP protein inLenti-GFP-infected cells with a dose-dependent effect. Treatment withCompound I-1 2 μM also showed an inhibition effect (FIG. 28(c)). Theseresults indicate that the benzopyran-4-one derivative compounds areeffective in treating retrovirus infection. Compound II-1 is more potentin treating retrovirus infection than Compound I-1.

Based on the treatment protocol shown in FIG. 26, pre-treatment ofbenzopyran-4-one derivative compounds was more efficient thanpost-treatment. We infer that benzopyran-4-one derivative compoundsaffect virus infection in multiple steps. Moreover, the inhibitionpatterns of benzopyran-4-one derivative compounds in Ad-GFP andLenti-GFP are quite similar, indicating that the mechanisms of thesecompounds for DNA virus and retrovirus infections are similar.

Ideally, the Ad-GFP virus is replicated within HEK293A cells. To testthe effect of benzopyran-4-one derivative compounds on Ad-GFP virusreplication, the same virus infection and compound treatment areperformed as shown in FIG. 26. At the end of a 24-hour experiment, wecollected the Ad-GFP virus from each well of Ad-GFP infected HEK293Ausing two freeze-thaw cycles and then collected the supernatants. Anequal amount of supernatants from each well were then infected with theHEK293A cells, which were newly-prepared 1 day before infection in a96-well plate at a density of 5×10⁴ per well. Finally, the GFP cellimages were obtained using the inverted fluorescence microscope andquantified by analyzing the MFI using flow cytometry for each well ofcells.

24 hours after isolating the Ad-GFP virus infected with the new preparedHEK293A cells, we found that the pre-treatment with Compound I-1 orCompound II-1 significantly decreased the number of GFP cells with adose-dependent effect (FIG. 29(a)) as well as the MFI of GFP protein(FIG. 29(b)). The inhibition of virus infection including virusattaching to or entering the cells, may account for the inhibition ofvirus replication with Compound II-1 due to the significant inhibitionof virus infection (FIG. 27). However, Compound I-1 significantlyinhibited the virus replication in cells, proving that benzopyran-4-onederivative compounds prevent adenovirus replication since the same doseof Compound I-1 did not significantly inhibit the Ad-GFP virus infection(FIG. 27). Moreover, post-treatment of Compound I-1 or Compound II-1after 2 hours of Ad-GFP virus infection significant decreased the numberof GFP cells with a dose-dependent effect (FIG. 29(c)) as well as theMFI of GFP protein (FIG. 29(d)).

To understand whether or not inhibition of the virus infection isfollowed by the host cell toxicity caused by the compound treatments, weperformed an MTT [3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide] assay in cells untreated or treated with indicated compoundsfor 6 hours. After that, the compound is washed out and the MTT assaywas tested after an additional 18 hours of fresh culture mediumincubation, in a way similar to the protocol shown in FIG. 26. 0.5 mg/mLof MTT contained media was added to cells at the end of the experimentand incubated for 3 hours. Then, the supernatant was removed and theformazone crystals solubilized using 100 μl of DMSO and the absorbanceat 570 nm was read using a microplate reader. The result showed adecrease of 5-10% of surviving cells in the highest dose of 1 μMCompound II-1 (FIG. 30). However, the virus inhibition effect of 50-99%is also shown at this dose of Compound II-1. Therefore, our results showthat the compound effect shown by the MFI of GFP protein, which is areporter of virus infection, was not interfered with cell density in theexperiments.

Suitable dose ranges and cell toxicity levels may be assessed usingstandard dose range experiments that are well-known in the art. Actualdosages administered may vary depending, for example, on the nature ofthe disorder, e.g., stage of virus-mediated pathology, the age, weightand health of the individual, as well as other factors.

In some embodiments, the compounds in this disclosure are formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, pills, powders, granules, dragees, gels, slurries,ointments, solutions, suppositories, injections, inhalants and aerosols.Administration of such formulations can be achieved in various ways,including oral, buccal, parenteral, injection, intravenous, intradermal(e.g., subcutaneous, intramuscular), topical, transdermal, transmucosal,inhalation, nasal, rectal, vaginal, etc., administration. Moreover, thecompounds in this disclosure can be administered in a local rather thansystemic manner, for example, in a depot or sustained releaseformulation.

In some embodiments, a method for treating or preventing a virusinfection in a subject is disclosed. The method comprises a step ofadministering to the subject with the virus infection a therapeuticallyeffective amount of a benzopyran-4-one derivative for a administrationduration, i.e. the treatment duration, between 1 to 10 days to inhibit aviral replication in the subject. Some studies suggest that antiviraltreatment may be beneficial in hospitalized patients when started up to4 or 5 days after illness onset. Treatment duration might need to bealtered to fit the clinical circumstances. For example, clinicaljudgment should be the guide regarding the need to extend treatmentduration longer than 5 or 10 days for patients whose illness isprolonged. In some embodiments, the method comprises a step ofidentifying the subject with the virus infection. In some embodiments,the benzopyran-4-one derivative is administered intraperitoneally orsubcutaneously. In some embodiments, the virus is one of an adenovirusand a retrovirus. In some embodiments, the subject is at risk ofdeveloping a viral infection. In some embodiments, the benzopyran-4-onederivative is administered at an interval selected from a groupconsisting of a once-daily interval, a multiple-daily interval (onceevery 8 or 12 hrs) and a weekly interval. Dosage adjustment may beperformed to maximize efficacy and minimize toxicity. In patients withhigh clearance (e.g., young adults), dosing intervals shorter than 24hrs may be more appropriate. In some embodiments, the benzopyran-4-onederivative is administered in combination with at least one agentselected from a group consisting of an antiviral agent, an antibiotic,and a steroid drug. In some embodiments, the antiviral agent is ananti-retroviral agent selected from a group consisting of a nucleosidereverse transcriptase inhibitor, a nucleotide reverse transcriptaseinhibitor, a non-nucleoside reverse transcriptase inhibitor, a proteaseinhibitor, a fusion inhibitor, and an integrase inhibitor. In someembodiments, the virus infection is one of an adenovirus infection,which may be a respiratory infection, and a retrovirus infection.

REFERENCES

-   Andreassen P R, et al. ATR couples FANCD2 monoubiquitination to the    DNA-damage response. Genes Dev 2004; 18(16): 1958-63.-   Chen H M, et al. A novel synthetic protoapigenone analogue, WYC02-9,    induces DNA damage and apoptosis in DU145 prostate cancer cells    through generation of reactive oxygen species. Free Radic Biol Med    2011; 50(9): 1151-62.-   Chen W Y, et al. Protoapigenone, a natural derivative of apigenin,    induces mitogen-activated protein kinase-dependent apoptosis in    human breast cancer cells associated with induction of oxidative    stress and inhibition of glutathione S-transferase pi. Invest New    Drugs 2011; 29(6): 1347-59.-   Chirnomas D, et al. Chemosensitization to cisplatin by inhibitors of    the Fanconi anemia/BRCA pathway. Mol Cancer Ther 2006; 5(4): 952-61.-   Chiu C C, et al. Fern plant-derived protoapigenone leads to DNA    damage, apoptosis, and G(2)/m arrest in lung cancer cell line H1299.    DNA Cell Biol 2009; 28(10): 501-6.-   Lopez-Contreras A J, et al. The ATR barrier to replication-born DNA    damage. DNA Repair (Amst) 2010; 9(12): 1249-55.-   Nghiem P, et al. ATR inhibition selectively sensitizes G1    checkpoint-deficient cells to lethal premature chromatin    condensation. Proc Natl Acad Sci USA 2001; 98(16): 9092-7.-   Sorensen C S, et al. The cell-cycle checkpoint kinase Chk1 is    required for mammalian homologous recombination repair. Nat Cell    Biol 2005; 7(2): 195-201.-   Wang H C, et al. Inhibition of ATR-dependent signaling by    protoapigenone and its derivative sensitize cancer cells to    interstrand cross-link-generating agents in vitro and in vivo. Mol    Cancer Ther molcanther. Apr. 24, 2012; 1443

What is claimed is:
 1. A method for enhancing a chemosensitivity of aspecific chemotherapeutic drug, comprising a step of: administering atthe same time a therapeutically effective amount of a benzopyran-4-onederivative and the chemotherapeutic drug to a subject with a specificcancer, wherein the specific chemotherapeutic drug is a DNA damagingagent, and the therapeutically effective amount of the benzopyran-4-onederivative is to cause an effective blood concentration of thebenzopyran-4-one derivative in the subject in a range from 2.5 nM to 2μM.
 2. The method as claimed in claim 1, wherein the benzopyran-4-onederivative is represented by formula I:

wherein each of R₃, R₅, R₇, R₁₁, R₁₄ and R₁₆ is one selected from agroup consisting of a hydrogen, a hydroxyl group, a methoxyl group andan oxygen atom containing a double bond.
 3. The method as claimed inclaim 1, wherein the benzopyran-4-one derivative is represented byformula II:

wherein R₂₁ is one selected from a group consisting of a hydrogen, ahydroxy and a methoxyl group.
 4. The method as claimed in claim 1,wherein the DNA damaging agent is selected from a group consisting of aDNA-reactive agent, an antimetabolity, a topoisomerase poison and acombination thereof.
 5. The method as claimed in claim 4, wherein theDNA-reactive agent is selected from a group consisting of a nitrogenmustard, anitrosourea, a triazene, a natural resource alkylating agent,an alkylating-like platinum agent and a combination thereof.
 6. Themethod as claimed in claim 5, wherein the nitrogen mustard is selectedfrom a group consisting of DNA alkylators cyclophosphamide,chlorambucil, melphalan and a combination thereof; the nitrosoureas isselected from a group consisting of carmustine, lomustine, semustine anda combination thereof; the triazene is selected from a group consistingof dacarbazine, temozolomide and a combination thereof; the naturalresource alkylating agent is selected from a group consisting ofmitomycin C, streptozotocin and a combination thereof; and thealkylating-like platinum agent is selected from a group consisting ofcisplatin, carboplatin, oxaliplatin and a combination thereof.
 7. Themethod as claimed in claim 4, wherein the antimetabolity is selectedfrom a group consisting of a pyrimidine analog, a purine analog, anucleotide synthesis blocker and a combination thereof.
 8. The method asclaimed in claim 7, wherein the pyrimidine analog is selected from agroup consisting of 5-fluorouracil, capecitabine, floxuridine,gemcitabine and a combination thereof; the purine analog is selectedfrom a group consisting of 6-mercaptopurine, 8-azaguanine, fludarabine,cladribine and a combination thereof; and the nucleotide synthesisblocker is selected from a group consisting of methotrexate,aminopterin, pemetrexed, ralitrexed and a combination thereof.
 9. Themethod as claimed in claim 4, wherein the topoisomerase poison isselected from a group consisting of a topoisomerase II poison, atopoisomerase I poison, an anthracycline with the topoisomerase IIpoison plus a DNA intercalating property and a combination thereof. 10.The method as claimed in claim 9, wherein thetopoisomerase II poison isselected from a group consisting of etoposide, teniposide and acombination thereof; the topoisomerase I poison is camptothecin; and theanthracycline with the topoisomerase II poison plus the DNAintercalating property is selected from a group consisting ofdoxorubicin, daunorubicin, epirubicin, idarubicin and a combinationthereof.
 11. The method as claimed in claim 1, wherein the subject is amammal subject.
 12. The method as claimed in claim 11, wherein thechemosensitivity of the specific chemotherapeutic drug is enhancedthrough administering at the same time the benzopyran-4-one derivativeand the chemotherapeutic drug to treat the mammal subject who has one ofrecurrent cancer, a cancer having a cancer chemotherapy resistance, acancer having a resistance to DNA damage response (DDR) and acombination thereof.